EP3300591A1 - Xanthomonas resistant brassica oleracea plants - Google Patents

Xanthomonas resistant brassica oleracea plants Download PDF

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Publication number
EP3300591A1
EP3300591A1 EP17194031.5A EP17194031A EP3300591A1 EP 3300591 A1 EP3300591 A1 EP 3300591A1 EP 17194031 A EP17194031 A EP 17194031A EP 3300591 A1 EP3300591 A1 EP 3300591A1
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Prior art keywords
plant
resistance
brassica oleracea
introgression
campestris
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German (de)
English (en)
French (fr)
Inventor
Gerard N. Koorevaar
Carl E. Mero
Franciscus Van Den Bosch
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Seminis Vegetable Seeds Inc
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Seminis Vegetable Seeds Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A01H6/203—Brassica oleraceae, e.g. broccoli or kohlrabi
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/021—Methods of breeding using interspecific crosses, i.e. interspecies crosses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/125—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for bacterial resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance

Definitions

  • the present invention relates to the field of agriculture and more specifically to methods and compositions for producing Brassica oleracea plants exhibiting improved resistance to Xanthomonas campestris pv. campestris.
  • MAS marker-assisted selection
  • the present invention provides a cultivated plant line or variety of a Brassica oleracea plant comprising an introgression from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv. campestris relative to a plant lacking the introgression, and wherein the resistance is co-dominant and additive.
  • the introgression lacks an allele genetically linked thereto conferring retarded plant growth.
  • the introgression comprises a chromosomal segment from Brassica oleracea var. capitata flanked by marker NH0265157 (SEQ ID NO:5) and marker NH0266951 (SEQ ID NO:12) in the plant.
  • the introgression comprises a chromosomal segment from Brassica oleracea var. capitata at marker NH0265597 (SEQ ID NO:11) in the plant.
  • the introgression is about 2 cM or less in size.
  • the plant is homozygous for the introgression.
  • a sample of seed comprising the introgression was deposited under ATCC Accession Number PTA-123409.
  • the broad-spectrum resistance comprises resistance to a plurality of Xanthomonas campestris pv. campestris pathovars.
  • the broad-spectrum resistance comprises resistance to Xanthomonas campestris pv. campestris pathovars 1 and 4.
  • the broad-spectrum resistance comprises resistance to Xanthomonas campestris pv. campestris pathovars 1, 4, 6 and 9.
  • the plant is a broccoli, cauliflower, sprouting broccoli, Brussels sprouts, white cabbage, red cabbage, savoy cabbage, curly kale cabbage, turnip cabbage or Portuguese cabbage plant.
  • the plant is not a white cabbage plant.
  • the present invention additionally provides a seed that produces a cultivated plant line or variety of a Brassica oleracea plant comprising an introgression from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv. campestris relative to a plant lacking the introgression, and wherein the resistance is co-dominant and additive.
  • the present invention also provides a plant part from a cultivated plant line or variety of a Brassica oleracea plant comprising an introgression from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv.
  • the plant part is a cell, a seed, a root, a stem, a leaf, a head, a flower, or pollen.
  • the present invention provides an introgression fragment comprising a chromosomal segment from Brassica oleracea var. capitata flanked by marker NH0265157 (SEQ ID NO:5) and marker NH0266951 (SEQ ID NO:12), an introgression fragment comprising a chromosomal segment from Brassica oleracea var. capitata at marker NH0265597 (SEQ ID NO:11), and an introgression fragment comprising a chromosomal segment from Brassica oleracea var. capitata wherein the fragment confers broad-spectrum resistance to Xanthomonas campestris pv. campestris and lacks an allele genetically linked thereto conferring retarded plant growth.
  • the present invention also provides a broccoli or cauliflower plant comprising an introgression from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv. campestris relative to a plant lacking the introgression, and wherein the resistance is co-dominant and additive.
  • the present invention further provides a method for producing a cultivated plant line or variety of a Brassica oleracea plant with improved resistance to Xanthomonas campestris pv. campestris, comprising introgressing into the plant plant line or variety a chromosomal segment from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv. campestris relative to a plant lacking the introgression, and wherein the resistance is co-dominant and additive.
  • the introgressing comprises crossing a plant comprising the chromosomal segment with itself or with a second Brassica oleracea plant of a different genotype to produce one or more progeny plants, and selecting a progeny plant comprising the chromosomal segment.
  • selecting a progeny plant comprises detecting at least one allele flanked by marker NH0265157 (SEQ ID NO:5) and marker NH0266951 (SEQ ID NO:12).
  • selecting a progeny plant comprises detecting at least one allele at marker NH0265597 (SEQ ID NO:11).
  • the progeny plant is an F2-F6 progeny plant.
  • the crossing comprises backcrossing. In additional embodiments the backcrossing comprises from 2-7 generations of backcrosses.
  • the present invention further provides a method for selecting a cultivated plant line of a Brassica oleracea plant with improved resistance to Xanthomonas campestris pv. campestris, which method comprises introgressing into said plant line a chromosomal segment from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv. campestris relative to a plant lacking said introgression, and wherein said resistance is co-dominant and additive.
  • the plant plant line or variety is a broccoli, cauliflower, sprouting broccoli, Brussels sprouts, white cabbage, red cabbage, savoy cabbage, curly kale cabbage, turnip cabbage or Portuguese cabbage plant variety.
  • the resistance comprises resistance to a plurality of Xanthomonas campestris pv. campestris pathovars.
  • the resistance comprises resistance to Xanthomonas campestris pv. campestris pathovars 1 and 4.
  • the resistance comprises resistance to Xanthomonas campestris pv. campestris pathovars 1, 4, 6 and 9.
  • a sample of seed comprising the chromosomal segment was deposited under ATCC Accession Number PTA-123409.
  • the present invention further provides a cultivated plant line or variety of a Brassica oleracea plant with improved resistance to Xanthomonas campestris pv. campestris, produced by introgressing into the plant plant line or variety a chromosomal segment from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv. campestris relative to a plant lacking the introgression, and wherein the resistance is co-dominant and additive.
  • the present invention also provides a method of producing food or feed comprising obtaining a Brassica oleracea plant, or a part thereof, with improved resistance to Xanthomonas campestris pv.
  • campestris that comprises an introgressed chromosomal segment from Brassica oleracea var. capitata chromosome 3 that confers broad-spectrum resistance to Xanthomonas campestris pv. campestris relative to a plant lacking the introgression, wherein the resistance is co-dominant and additive, and producing the food or feed from the plant or part thereof.
  • Black rot is a bacterial disease of crucifers caused by Xanthomonas campestris pv. campestris (Xcc).
  • Xcc is transmitted by direct infection of developing seeds.
  • Xcc is present across a wide geographical area and particularly affects cultivated plant lines or varieties of Brassica oleracea by reducing yield and quality.
  • Black rot or Xcc causes significant economic damage in cultivated plant lines or varieties of Brassica oleracea, including, but not limited to, Brussels sprouts, cabbage, cauliflower, broccoli, and kale.
  • pathovars or races of Xanthomonas campestris pv. campestris has been undertaken by various groups with some variation in the classification and numbers of races or pathovars. At least 7 pathovars of Xcc have been identified Vicente, et al., Phytopathology 91:492-499, 2001 ; Fargier and Manceau, Plant Pathology 56:805-818, 2007 ; Jensen, et al., Plant Disease 94:298-305, 2010 . These pathovars will often co-occur with one dominant pathovar. Races 1 and 4 are considered of major importance.
  • a resistance allele preferably a single gene, and for the allele to provide resistance to more than two races of Xcc.
  • the present invention represents a significant advance in that it provides a single resistance QTL that provides resistance to at least two pathovars of Xcc, and more particularly to at least pathovars 1 and 4.
  • This QTL can be introgressed into elite lines of cultivated plant lines or varieties of Brassica oleracea. These plant lines or varieties can include, but are not limited to, cultivated plant lines or varieties of broccoli, cauliflower, sprouting broccoli, Brussels sprouts, white cabbage, red cabbage, savoy cabbage, curly kale cabbage, turnip cabbage and Portuguese cabbage.
  • the current invention provides a resistant locus without an associated allele that confers retarded growth relative to a plant that lacks the associated allele. Such retarded growth may include a slower rate of plant growth, as well as a reduced overall mature plant size.
  • Brassica oleracea hosts have been identified that are resistant to multiple Xcc isolates (Taylor, 2002), these resistances have been found to be limited to the less common races and none have provided resistance to both races 1 and 4. In other cases, the resistance was found to be partial and quantitative ( Lema, et al., Plant Breeding 131:607-613, 2012 (“Lema, 2012”)). The race non-specific resistances depend on the combined action of various genes each of which has a small effect. These resistances are therefore hard to manage and transfer between cultivars. Most of the publications have concluded that the resistance follows a gene-for-gene model and that the resistance provided is race specific. Each different resistance gene was generally believed to provide resistance against one pathovar. Saha, et al. (Plant Breeding 133:268-274, 2014 ) identified a single dominant resistance QTL from a cauliflower source. However, this was shown to have resistance only to race 1.
  • the single resistance QTL of the present invention was identified on chromosome 3 (O3).
  • the initial resistance allele identified was found to be tightly associated with a retarded growth plant type locus.
  • the present invention also provides a smaller introgression fragment, which breaks the linkage. It was surprisingly found that a recombined chromosomal fragment could be generated that lacks the deleterious retarded growth plant type.
  • the recombined introgression fragment was identified using marker assisted breeding techniques and the introgression fragment generated had a size of about 2 centiMorgans (cM). The mapping of this chromosomal segment found that the QTL for Xanthomonas resistance is located at marker NH0265597 (76.45 cM).
  • the QTL is flanked by markers NH0265157 (74.98 cM) and NH0266951 (77.14 cM). This QTL results in resistance to several different Xcc pathovars, including pathovars 1 and 4, without the deleterious retarded growth plant type.
  • the resistance based on the recombined introgression is additive. Therefore, it is preferable for the QTL to be homozygous in hybrid plants, particularly for commercial hybrids. It is also generally preferable to use the smallest introgression fragment comprising the Xanthomonas resistance since the homozygous presence of other associated alleles can cause expression of deleterious recessive traits.
  • the recombined introgression provided by the present invention has been shown to provide resistance to a plant line or variety of different pathovars, including the currently prevalent pathovars 1, 4, 6 and 7, along with several unclassified isolates.
  • the resistance QTL was tested against a group of 157 Xcc isolates from Brassica oleracea crops (broccoli, cauliflower, cabbage) that were identified from areas around the world.
  • the isolates were classified using the improved differential plant series of Vicente, 2001, which is the current standard for the scientific community, and most of the isolates could be classified into pathovar groups 1, 4, 6 and 9. However, some of the isolates could not be classified.
  • the differential set of Vicente consists of different Brassica plant species and enables pathovar differentiation based on compatible (susceptibility) and incompatible (resistance) interactions. This set can therefore be used to phenotypically confirm the broad-spectrum disease resistance conferred by the invention.
  • Brassica is a plant genus of the family of brassicaceae (formerly referred to as cruciferae ). The members of this genus are also known as cabbage or mustard.
  • the genus Brassica comprises a number of commercially and agriculturally important species. Of all those species Brassica oleracea is the most diverse containing at least ten different commercial cultivated plant lines or varieties, including broccoli, cauliflower, sprouting broccoli, Brussels sprouts, white cabbage, red cabbage, savoy cabbage, curly kale cabbage, turnip cabbage and Portuguese cabbage. Breeding between these types is common and easily done because these types, while highly diverse phenotypically, are the same species, which means that a cross between the different types can be made without having to overcome any genetic species barrier.
  • Black rot is a bacterial disease in Brassica oleracea caused by Xanthomonas campestris pv. campestris (Xcc). Although some sources of black rot resistance have been identified, this resistance has not been successfully transferred to cultivated plant lines or varieties of Brassica oleracea, and in particular to broccoli and cauliflower. In addition, a single, co-dominant, additive QTL has not been previously identified.
  • Applicants were surprisingly able to identify a novel Xcc resistance region from Brassica oleracea var. capitata, identified in a cross between the publicly known cabbage line C517 and an elite broccoli line (BRL51-99sc).
  • the mapping of this introgression indicated that the QTL for Xcc resistance is located at marker NH0265597 (76.45 cM), and flanked by markers NH0265157 (74.98 cM) and NH0266951 (77.14 cM).
  • interval values may vary based on factors such as the reference map that is used, the sequencing coverage and the assembly software settings.
  • Marker-assisted introgression involves the transfer of a chromosomal region defined by one or more markers from a first genetic background to a second. Offspring of a cross that contain the introgressed genomic region can be identified by the combination of markers characteristic of the desired introgressed genomic region from a first genetic background and both linked and unlinked markers characteristic of the second genetic background.
  • the present invention provides novel markers for identifying and tracking introgression of one or more of the genomic regions from Brassica oleracea var. capitata, initially identified in a cross between cabbage line C517 (public source) and an elite broccoli line (BRL51-99sc), disclosed herein into cultivated Brassica oleracea lines.
  • the invention further provides markers for identifying and tracking the novel introgressions disclosed herein during plant breeding, including markers NH0265597 (76.45 cM), NH0265157 (74.98 cM) and NH0266951 (77.14 cM).
  • Markers within or linked to any of the genomic intervals of the present invention can be used in a plant line or variety of breeding efforts that include introgression of genomic regions associated with disease resistance into a desired genetic background.
  • a marker within 20 cM, 15 cM, 10 cM, 5cM, 2 cM, or 1 cM of a marker associated with disease resistance described herein can be used for marker-assisted introgression of genomic regions associated with a disease tolerant phenotype.
  • Brassica oleracea plants comprising one or more introgressed regions associated with a desired phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or 99% of the remaining genomic sequences carry markers characteristic of the germplasm are also provided. Brassica oleracea plants comprising an introgressed region comprising regions closely linked to or adjacent to the genomic regions and markers provided herein and associated with an Xcc disease resistance phenotype are also provided.
  • cultivars For most breeding objectives, commercial breeders work within germplasm that is "cultivated type” or “elite.” This germplasm is easier to breed because it generally performs well when evaluated for horticultural performance. Numerous elite Brassica oleracea crop cultivated plant lines or varieties (cultivars) have been developed, including, but not limited to, broccoli, cauliflower, sprouting broccoli, Brussels sprouts, white cabbage, red cabbage, savoy cabbage, curly kale cabbage, turnip cabbage and Portuguese cabbage. However, the performance advantage a cultivated or elite germplasm provides can be offset by a lack of allelic diversity. Breeders generally accept this tradeoff because progress is faster when working with cultivated material than when breeding with genetically diverse sources.
  • the alleles, polymorphisms, and markers provided by the invention allow the tracking and introduction of any of the genomic regions identified herein into any genetic background to which a Brassica oleracea species can be crossed.
  • the genomic regions associated with disease resistance disclosed herein can be introgressed from one genotype to another and tracked phenotypically or genetically.
  • Applicants' development markers for the selection of the disease resistance facilitates the development of Brassica oleracea plants having beneficial phenotypes.
  • plants and seeds can be genotyped using the markers of the present invention in order to develop plant lines or varieties comprising desired disease resistance.
  • marker-assisted selection allows identification of plants which are homozygous or heterozygous the desired introgression.
  • Meiotic recombination is essential for plant breeding because it enables the transfer of favorable alleles across genetic backgrounds, the removal of deleterious genomic fragments, and pyramiding traits that are genetically tightly linked. In the absence of accurate markers, limited recombination forces breeders to enlarge segregating populations for progeny screens. Moreover, phenotypic evaluation is time-consuming, resource-intensive and not reproducible in every environment, particularly for traits like disease resistance.
  • the markers provided by the invention offer an effective alternative and therefore represent a significant advance in the art.
  • Genetic markers that can be used in the practice of the present invention include, but are not limited to, restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), simple sequence length polymorphisms (SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (Indels), variable number tandem repeats (VNTRs), and random amplified polymorphic DNA (RAPD), isozymes, and other markers known to those skilled in the art.
  • RFLPs restriction fragment length polymorphisms
  • AFLPs amplified fragment length polymorphisms
  • SSRs simple sequence repeats
  • SSLPs simple sequence length polymorphisms
  • SNPs single nucleotide polymorphisms
  • Indels insertion/deletion polymorphisms
  • VNTRs variable number tandem repeats
  • RAPD random amplified polymorphic DNA
  • Advanced marker technologies are based on genome sequences, the nucleotide order of distinct, polymorphic genotypes within a species. Such platforms enable selection for horticultural traits with markers linked to favorable alleles, in addition to the organization of germplasm using markers randomly distributed throughout the genome.
  • sequence homology rather than known polymorphisms, to develop marker platforms.
  • Man-made DNA molecules are used to prime replication of genome fragments when hybridized pair-wise in the presence of a DNA polymerase enzyme.
  • RFLP RFLP does not include a DNA amplification step.
  • Amplified fragment length polymorphism (AFLP) technology reduces the complexity of the genome. First, through digestive enzymes cleaving DNA strands in a sequence-specific manner. Fragments are then selected for their size and finally replicated using selective oligonucleotides, each homologous to a subset of genome fragments. As a result, AFLP technology consistently amplifies DNA fragments across genotypes, experiments and laboratories.
  • Polymorphisms comprising as little as a single nucleotide change can be assayed in a number of ways. For example, detection can be made by electrophoretic techniques including a single strand conformational polymorphism ( Orita, et al., Genomics 8:271-278, 1989 ), denaturing gradient gel electrophoresis ( Myers, EP 0273085 ), or cleavage fragment length polymorphisms (Life Technologies, Inc., Gathersberg, MD), but the widespread availability of DNA sequencing often makes it easier to simply sequence amplified products directly.
  • electrophoretic techniques including a single strand conformational polymorphism ( Orita, et al., Genomics 8:271-278, 1989 ), denaturing gradient gel electrophoresis ( Myers, EP 0273085 ), or cleavage fragment length polymorphisms (Life Technologies, Inc., Gathersberg, MD), but the widespread availability of DNA sequencing often makes it easier to simply sequence amplified products directly.
  • Polymorphic markers serve as useful tools for assaying plants for determining the degree of identity of lines or varieties ( U.S. Patent No. 6,207,367 ). These markers form the basis for determining associations with phenotypes and can be used to drive genetic gain.
  • polymorphic nucleic acids can be used to detect in a Brassica oleracea plant a genotype associated with disease resistance, identify a Brassica oleracea plant with a genotype associated with disease resistance, and to select a Brassica oleracea plant with a genotype associated with disease resistance.
  • polymorphic nucleic acids can be used to produce a Brassica oleracea plant that comprises in its genome an introgressed locus associated with disease resistance. In certain embodiments of the invention, polymorphic nucleic acids can be used to breed progeny Brassica oleracea plants comprising a locus associated with disease resistance.
  • Genetic markers may include "dominant” or “codominant” markers. "Codominant” markers reveal the presence of two or more alleles (two per diploid individual). "Dominant” markers reveal the presence of only a single allele. Markers are preferably inherited in codominant fashion so that the presence of both alleles at a diploid locus, or multiple alleles in triploid or tetraploid loci, are readily detectable, and they are free of environmental variation, i.e., their heritability is 1.
  • a marker genotype typically comprises two marker alleles at each locus in a diploid organism.
  • the marker allelic composition of each locus can be either homozygous or heterozygous. Homozygosity is a condition where both alleles at a locus are characterized by the same nucleotide sequence. Heterozygosity refers to different conditions of the allele at a locus.
  • Nucleic acid-based analyses for determining the presence or absence of the genetic polymorphism can be used in breeding programs for identification, selection, introgression, and the like.
  • a wide variety of genetic markers for the analysis of genetic polymorphisms are available and known to those of skill in the art. The analysis may be used to select for genes, portions of genes, QTL, alleles, or genomic regions that comprise or are linked to a genetic marker that is linked to or associated with disease resistance in Brassica oleracea plants.
  • nucleic acid analysis methods include, but are not limited to, PCR-based detection methods (for example, TaqMan assays), microarray methods, mass spectrometry-based methods and/or nucleic acid sequencing methods, including whole genome sequencing.
  • the detection of polymorphic sites in a sample of DNA, RNA, or cDNA may be facilitated through the use of nucleic acid amplification methods.
  • Such methods specifically increase the concentration of polynucleotides that span the polymorphic site, or include that site and sequences located either distal or proximal to it.
  • Such amplified molecules can be readily detected by gel electrophoresis, fluorescence detection methods, or other means.
  • PCR polymerase chain reaction
  • Polymorphisms in DNA sequences can be detected or typed by a variety of effective methods well known in the art including, but not limited to, those disclosed in U.S. Patent Nos. 5,468,613 , 5,217,863 ; 5,210,015 ; 5,876,930 ; 6,030,787 ; 6,004,744 ; 6,013,431 ; 5,595,890 ; 5,762,876 ; 5,945,283 ; 5,468,613 ; 6,090,558 ; 5,800,944 ; 5,616,464 ; 7,312,039 ; 7,238,476 ; 7,297,485 ; 7,282,355 ; 7,270,981 and 7,250,252 all of which are incorporated herein by reference in their entirety.
  • compositions and methods of the present invention can be used in conjunction with any polymorphism typing method to type polymorphisms in genomic DNA samples.
  • genomic DNA samples used include but are not limited to, genomic DNA isolated directly from a plant, cloned genomic DNA, or amplified genomic DNA.
  • polymorphisms in DNA sequences can be detected by hybridization to allele-specific oligonucleotide (ASO) probes as disclosed in U.S. Patent Nos. 5,468,613 and 5,217,863 .
  • ASO allele-specific oligonucleotide
  • U.S. Patent No. 5,468,613 discloses allele specific oligonucleotide hybridizations where single or multiple nucleotide variations in nucleic acid sequence can be detected in nucleic acids by a process in which the sequence containing the nucleotide variation is amplified, spotted on a membrane and treated with a labeled sequence-specific oligonucleotide probe.
  • Target nucleic acid sequence can also be detected by probe ligation methods, for example as disclosed in U.S. Patent No. 5,800,944 where sequence of interest is amplified and hybridized to probes followed by ligation to detect a labeled part of the probe.
  • Microarrays can also be used for polymorphism detection, wherein oligonucleotide probe sets are assembled in an overlapping fashion to represent a single sequence such that a difference in the target sequence at one point would result in partial probe hybridization ( Borevitz, et al., Genome Res. 13:513-523, 2003 ; Cui, et al., Bioinformatics 21:3852-3858, 2005 ).
  • target sequences which may represent genes and/or noncoding regions wherein each target sequence is represented by a series of overlapping oligonucleotides, rather than by a single probe.
  • This platform provides for high throughput screening of a plurality of polymorphisms. Typing of target sequences by microarray-based methods is disclosed in US Patent Nos. 6,799,122 ; 6,913,879 ; and 6,996,476 .
  • SBE single base extension
  • SNPs and Indels can be detected by methods disclosed in U.S. Patent Nos. 5,210,015 ; 5,876,930 ; and 6,030,787 in which an oligonucleotide probe having a 5' fluorescent reporter dye and a 3' quencher dye covalently linked to the 5' and 3' ends of the probe.
  • an oligonucleotide probe having a 5' fluorescent reporter dye and a 3' quencher dye covalently linked to the 5' and 3' ends of the probe.
  • the proximity of the reporter dye to the quencher dye results in the suppression of the reporter dye fluorescence, e.g. by Forster-type energy transfer.
  • a locus or loci of interest can be directly sequenced using nucleic acid sequencing technologies.
  • Methods for nucleic acid sequencing are known in the art and include technologies provided by 454 Life Sciences (Branford, CT), Agencourt Bioscience (Beverly, MA), Applied Biosystems (Foster City, CA), LI-COR Biosciences (Lincoln, NE), NimbleGen Systems (Madison, WI), Illumina (San Diego, CA), and VisiGen Biotechnologies (Houston, TX).
  • nucleic acid sequencing technologies comprise formats such as parallel bead arrays, sequencing by ligation, capillary electrophoresis, electronic microchips, "biochips,” microarrays, parallel microchips, and single-molecule arrays.
  • plant includes plant cells, plant protoplasts, plant cells of tissue culture from which Brassica oleracea plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants such as pollen, flowers, seeds, leaves, stems, and the like.
  • population means a genetically heterogeneous collection of plants that share a common parental derivation.
  • plant line refers to a group of similar plants not characterized by genetic pedigrees and performance which can be identified from other varieties within the same species.
  • plant line encompasses a group of varieties and a group of similar plants that are sufficiently fixed in their genetic pedigree that they can be considered as a unit with regard to its suitability for being propagated unchanged.
  • an "allele” refers to one of two or more alternative forms of a genomic sequence at a given locus on a chromosome.
  • QTL Quality of Trait Locus
  • a "marker” means a detectable characteristic that can be used to discriminate between organisms. Examples of such characteristics include, but are not limited to, genetic markers, biochemical markers, metabolites, morphological characteristics, and agronomic characteristics.
  • phenotype means the detectable characteristics of a cell or organism that can be influenced by gene expression.
  • the term "genotype" means the specific allelic makeup of a plant.
  • elite line or “cultivated line” means any line that has resulted from breeding and selection for superior agronomic performance.
  • An “elite plant” refers to a plant belonging to an elite line. Numerous elite lines are available and known to those of skill in the art of Brassica oleracea breeding.
  • An “elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as a Brassica oleracea line.
  • an "elite germplasm” or elite strain of germplasm is an agronomically superior germplasm.
  • a cultivated variety of Brassica oleracea is not intended to encompass the C517 white cabbage source.
  • Introgressed when used in reference to a genetic locus, refers to a genetic locus that has been introduced into a new genetic background, such as through backcrossing. Introgression of a genetic locus can be achieved through plant breeding methods and/or by molecular genetic methods. Such molecular genetic methods include, but are not limited to, marker assisted selection.
  • the term "linked,” when used in the context of nucleic acid markers and/or genomic regions, means that the markers and/or genomic regions are located on the same linkage group or chromosome such that they tend to segregate together at meiosis.
  • resistance locus means a locus associated with resistance or tolerance to disease.
  • a resistance locus according to the present invention may, in one embodiment, control resistance or susceptibility to black rot.
  • resistance allele means the nucleic acid sequence associated with resistance or tolerance to disease.
  • resistance or “improved resistance” in a plant to disease conditions is an indication that the plant is less affected by disease conditions with respect to yield, survivability and/or other relevant agronomic measures, compared to a less resistant, more "susceptible” plant. Resistance is a relative term, indicating that a "resistant” plant survives and/or produces better yields in disease conditions compared to a different (less resistant) plant grown in similar disease conditions. As used in the art, disease “tolerance” is sometimes used interchangeably with disease “resistance.”
  • plant resistance to disease conditions varies widely, and can represent a spectrum of more-resistant or less-resistant phenotypes. However, by simple observation, one of skill can generally determine the relative resistance or susceptibility of different plants, plant lines or plant families under disease conditions, and furthermore, will also recognize the phenotypic gradations of "resistant.”
  • any forms or tenses of one or more of these verbs are also open-ended.
  • any method that "comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
  • any plant that "comprises,” “has” or “includes” one or more traits is not limited to possessing only those one or more traits and covers other unlisted traits.
  • a deposit was made of at least 2500 seeds of cauliflower strain C517xBRL51-99, which comprises an introgression from Brassica oleracea var. capitata, as described herein.
  • the deposit was made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA.
  • the deposit is assigned ATCC Accession No. PTA-123409, and the date of deposit was August 4, 2016. Access to the deposit will be available during the pendency of the application to persons entitled thereto upon request.
  • the deposit will be maintained in the ATCC Depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if nonviable during that period. Applicant does not waive any infringement of their rights granted under this patent or any other form of variety protection, including the Plant Variety Protection Act (7 U.S.C. 2321 et seq. ) .
  • DH Doubled Haploid
  • a suspension of Xanthamonas campestris campestris at a concentration of 1 x 10 6 cells/mL is used to inoculate the seedlings.
  • the seedlings are infected with the bacteria by injecting 0.2 mL of the Xcc suspension into the stem at two different points using a syringe.
  • the response of the seedlings is recorded when controls are starting to show the expected reactions, which generally is around 14 days post infection. Symptoms are scored on a scale of 1-9 with 1 being no symptoms (resistant) and 9 being many necrotic spots and/or death of the plants (susceptible).
  • Intermediate phenotypes are: 2: about two small spots visible on whole plant (total impression of plant is resistant); 3: very few chlorotic spots on the leaves (total impression of plant is resistant); 4: chlorotic spots on about 30% of leaf surface and whole plant (total impression of plant is IR); 5: chlorotic spots on about 40% of the leaf surface (total impression is IR), 6: chlorotic and necrotic spots on about 50% of leaves and whole plant (IR); 7: chlorotic and necrotic spots on about 60% of the leaf surface (susceptible); 8: necrotic spots on more than 70% of the leaf surface and whole plant (susceptible).
  • Each experiment will include controls to compare against the tested seedlings. These controls may include the original parents of the mapping population.
  • a molecular marker map was constructed using SSR loci. Combining the map and the quantitative disease data resulted in the detection of one major QTL for black rot resistance at chromosome O3 ( Figure 2 ).
  • 157 Xcc isolates were collected from Brassica oleracea crops (broccoli, cauliflower, cabbage) from all over the world. In this collection, isolates were classified using the improved differential plant series (Vicente, 2001), mostly as pathovar 1, 4, 6 and 9. Some isolates could not be classified into a pathovar group according to the Vicente, 2001 method. The distribution of pathovars across the world was not geographically linked, which corresponds to previous findings (Vicente, 2001; Taylor, 2002). All major pathovars were found to be present on every continent. This suggests that there is variation in Xcc for all growing areas worldwide. All isolates were then tested against the resistance QTL following the protocol described in Example 1. It was found that the resistance QTL provided resistance against all tested isolates (Table 1). Table 1.
  • the plants were then screened for six markers as indicated in Figure 2 which are positioned around the QTL.
  • Four (4) plants obtained from plots which were not segregating for retarded growth were sampled and all plants for plots segregating for retarded growth were sampled.
  • the retarded growth trait is highly correlated with the resistance QTL from markers NH0267115 to NH0267108 and appears to be recessive (Table 2).
  • Recombinants with a homozygous O3 introgression from NH0267115 to sN10957 and recurrent parent (HP99) for NH0265157 and NH0267108 do not show retarded growth.
  • the mapping of this introgression found that the QTL for Xanthomonas resistance is at most 2 cM in size and is located at marker NH0265597 (76.45 cM). Furthermore, the QTL is flanked by markers NH0265157 (74.98 cM) and NH0266951 (77.14 cM).
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP3607819A1 (en) * 2018-08-10 2020-02-12 Vilmorin et Cie Resistance to xanthomonas campestris pv. campestris (xcc) in cauliflower
WO2020030804A1 (en) * 2018-08-10 2020-02-13 Vilmorin & Cie Resistance to xanthomonas campestris pv. campestris (xcc) in cauliflower

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